Culture conditions and growth factors affecting fate determination, self-renewal and expansion of rat spermatogonial stem cells

a growth factor and fate determination technology, applied in cell culture active agents, instruments, dead animals, etc., can solve the problems of inability to examine the expression of thy-i on sscs in neonate or pup testis, elusive mechanisms underlying the process of self-renewal and differentiation of sscs, and inability to eliminate inhibitory testis somatic cells.

Inactive Publication Date: 2007-08-02
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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  • Abstract
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  • Claims
  • Application Information

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Benefits of technology

[0027] In another embodiment, the invention features a method of maintaining at least one SSC in a serum-free culture system, including providing a culture system comprising serum-free defined culture medium and mitotically-inactivated STO feeder cells, adding at least one enriched SSC to the culture system, and essentially eliminating inhibitory testis somatic cells and germ cells from the culture system.
[0028] The invention also features a method of proliferating at least one SSC in a serum-free culture system, comprising providing a culture system comprising serum-free defined culture medium and mitotically-inactivated STO feeder cells, and adding at least one enriched SSC to said culture system. In another embodiment, the invention features a method of proliferating at least one SSC in a serum-free culture system, the method comprising providing a culture system comprising serum-free defined culture medium and mitotically-inactivated STO feeder cells, adding at least one enriched SSC to the culture system, and essentially eliminating inhibitory testis somatic cells and germ cells from the culture system.

Problems solved by technology

Although SSCs and the surrounding microenvironment have been studied during the past decade using the transplantation assay (Brinster et al., Science, 296:2174-6 (2002)), mechanisms underlying the process of self-renewal and differentiation of SSCs remain elusive.
A major challenge still remaining is to establish a defined serum-free culture condition that supports maintenance of the stem cell and allows definitive experiments to analyze the effect of individual medium modifications on proliferation.
However, since crude cryptorchid testis cell populations were used as a stem cell source in the study, it is not clear whether STO cell feeders alone provide the beneficial effects on SSCs survival or the combination of STO cells and testis cells was necessary.
However, the expression of Thy-i on SSCs in neonate or pup testis has not been examined.

Method used

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  • Culture conditions and growth factors affecting fate determination, self-renewal and expansion of rat spermatogonial stem cells
  • Culture conditions and growth factors affecting fate determination, self-renewal and expansion of rat spermatogonial stem cells
  • Culture conditions and growth factors affecting fate determination, self-renewal and expansion of rat spermatogonial stem cells

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experimental examples

[0204] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

experimental example 1

Enrichment of Spermatogonial Stem Cells using Thy-1 and Establishment of Non-Serum Culture Conditions for Spermatogonial Stem Cells Donor Mice and Cell Collection

[0205] Cryptorchid and wild type adult donor testis cells were obtained from the transgenic mouse line B6.129S7-Gtrosa26 (designated ROSA; The Jackson Laboratory, Bar Habor, ME) that expresses the Escherichia coli LacZ gene in virtually all cell types, including all stages of spermatogenesis (Nagano et al., APMIS, 106:47-55 (1998)). Neonate (0.5-1.5 days postpartum, dpp; day of birth is 0.5 dpp), and pup (4.5-5.5 dpp) testis cells were collected from the hemizygous transgenic mice, C57BL / 6 x ROSA FI hybrid. Experimental cryptorchid testes were produced as previously described (Shinohara et al., Dev. Biol., 220:401-11 (2000)). Cell suspensions from cryptorchid adult, wild type adult, neonate, and pup testes were prepared by enzymatic digestion (Ogawa et al., Int. J. Dev. Biol., 41:111-22 (1997)). In several experiments, te...

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Abstract

The present invention relates to methods of identifying and enriching spermatogonioal stem cells, and compositions thereof. Further, the invention relates to methods and compositions for the isolation, maintenance and proliferation of spermatogonial stem cells, as well as methods and compositions for the identification and use of factors influencing spermatogonial stem cell maintenance and proliferation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of PCT International Application No. PCT / US2005 / 12273, filed Apr. 11, 2005, which in turn claims the benefit pursuant to 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60 / 561,464, filed Apr. 12, 2004 and 60 / 598,148, filed Aug. 2, 2004, all of which are hereby incorporated by reference in their entirety herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was supported in part by US Government funds (National Institutes of Health grant No. RO1 HD044445), and the US Government may therefore have certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Mammalian spermatogonial stem cells (SSCs) self-renew and produce daughter cells that commit to differentiate into spermatozoa throughout adult life of the male (Meistrich et al., Oxford Univ. Press; 266-295 (1993)). SSCs can be identified unequivocally by a functional assay using a transplantation t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A01N1/02G01N33/567C12N5/00C12N5/076C12N15/87
CPCA01K67/0271C12N5/061C12N5/0612C12N15/87C12N2500/99A01N1/0231C12N2501/115C12N2501/13G01N33/5073G01N33/56966A01N1/0226C12N2501/10C12N2500/90
Inventor BRINSTER, RALPH L.KUBOTA, HIROSHI
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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